1. Field of the Invention
The present invention relates to isolated peptides comprising agonist epitopes of prostate-specific antigen (PSA). In one aspect, this invention relates to peptides comprising agonist epitopes of the PSA-3 CTL (cytotoxic T lymphocyte) epitope. The invention also relates to nucleic acids (e.g., recombinant viral vectors) encoding peptides that comprise PSA-3 agonist epitopes, as well as host cells comprising these nucleic acids, and antibodies that bind to these peptides. Also related are pharmaceutical compositions comprising the nucleic acids and peptides of the invention, as well as methods of treatment of prostate cancer employing such compositions, e.g., peptide-mediated and vector-mediated immunotherapy.
2. Description of the Background
Current treatment for prostate cancer involves surgery, radiation, chemotherapy, and/or hormonal therapy. In spite of these treatments, over 40,000 men die of prostate cancer each year in the U.S. alone (C. C. Boring et al., 1994, CA Cancer J. Clin. 44:7–26). One promising modality for the treatment of prostate cancer involves vaccine therapy. Prostate-specific antigen (PSA), an antigen expressed on prostate carcinoma, is an important target for vaccine therapy (K. W. Watt et al., 1986, Proc. Natl. Acad. Sci. USA, 83:3166–3170). While PSA is expressed on normal prostatic epithelium, it is not expressed to any appreciable level on other normal adult tissues. However, PSA is a “self-antigen,” giving rise to immunotolerance.
Two vaccine clinical trials have been conducted using a recombinant vaccinia virus expressing the PSA gene (rV-PSA) as an immunogen. These studies demonstrated that both anti-PSA antibody responses (M. G. Sanda et al., 1999, Urology 53:260–266) and PSA-specific T cell responses (J. P. Eder et al., 2000, Clin. Cancer Res. 6:1632–1638) could be induced in vaccinated patients. In one trial, T cell responses to a 10-mer PSA peptide was induced in five out of seven prostate cancer patients following vaccination with rV-PSA. These results were achieved using an ELISPOT assay in which PBMCs of patients were incubated overnight with peptidepulsed APCs (antigen presenting cells). The short incubation period was used to prevent artifacts due to prolonged cycles of in vitro peptide stimulation of T cells (J. P. Eder et al., 2000, Clin. Cancer Res. 6:1632–1638). Notably, no PSA-specific T cell responses were observed when PBMCs were used prior to vaccination with rV-PSA.
The peptide used to monitor immune responses in patients receiving rV-PSA was an HLA-A2 binding 10-mer peptide, designated PSA-3 (VISNDVCAQV; SEQ ID NO:1; P. Correale et al., 1997, J. Natl. Cancer Inst. 19:293–300; P. Correale et al., 1998, J. Immunol. 161:3186–3194). The HLA-A2 binding peptide was used to monitor patients who were positive for the HLA-A2 allele. Patients possessing the HLA-A2 allele were chosen for monitoring because it is the most common HLA allele in individuals in North America and is expressed by approximately 50% of the Caucasian population (J. Lee, 1990, “The HLA system: a new approach” Proceedings Of The First Red Cross International Histocompatibility Workshop Springer Verlag, New York, p. 154).
Previous studies also demonstrated that the PSA-3 epitope was naturally processed by tumor cells, and bound to MHC-class I A2 molecules on the surface of prostate cancer cells. This binding rendered prostate cells susceptible to lysis by specific T cells in an MHC-restricted manner (P. Correale et al., 1998, J. Immunol. 161:3186–3194). In addition, prior studies analyzed the amino acid sequence of the PSA molecule using both computer algorithms, and by peptide-binding studies to the HLA-A2 positive T2A2 cell line (K. S. Anderson et al., 1993, J. Immunol. 151:3407–3419). Studies also analyzed the ability of various PSA peptides to generate CTL lines in vitro. This analysis demonstrated that the PSA-3 peptide was optimal for these properties.
The PSA-3 peptide was subsequently used in the ELISPOT assay to monitor immune responses in vaccinated patients (J. P. Eder et al., 2000, Clin. Cancer Res. 6:1632-1638; P. Arlen et al., 2000, Cancer Immuno. Immunother. 49:517–529). The results of these studies indicated that increases in precursor frequencies observed in the ELISPOT assay as a result of vaccination with rV-PSA were relatively modest (i.e., 2- to 4-fold) (J. P. Eder et al., 2000, Clin. Cancer Res. 6:1632–1638). Thus, there is a need in the art for compositions and methods for enhancing the immunogenicity of PSA.
The present invention therefore describes compositions and methods for increasing PSA immunogenicity, as well as treatments employing these compositions and methods. In accordance with this invention, the immunogenicity of PSA peptides was increased by modifying the amino acid residues that interact with HLA molecules. Previous studies have shown that amino acid modification in the anchor residues of peptides may result in enhanced binding to MHC and enhanced T cell activation, while other amino acid modifications will have no effect or act to antagonize T cell activation (H. M. Grey et al., 1995, Cancer Surv. 22:37–49; M. T. De Magistris et al., 1992, Cell 68:625–632; S. C. Jameson et al., 1995, Immunity 2:1–11; S. Zaremba et al., 1997, Cancer Res. 57:4570-4577). Enhancement of T cell activation by modifying HLA-anchor residues has been demonstrated for some human melanoma-associated antigens (D. Valmori et al., 1998., J. Immunol. 160:1750–1758, 1998; Y. Kawakami, Y., Eliyahu et al., 1995, J. Immunol. 154:3961–3968), but has not been demonstrated for antigens associated with most solid tumors, leukemias, or lymphomas.
The experiments of this invention, shown herein below, describe the design and analysis of an agonist of the PSA-3 CTL epitope, designated PSA-3A. These experiments demonstrate that when compared with the native PSA-3 epitope, the PSA-3A epitope exhibited enhanced binding to the MHC-class I A2 allele and resulted in enhanced stability of the peptide MHC complex. As shown herein, T cell lines generated with either the PSA-3 or the PSA-3A peptide showed higher levels of lysis of targets pulsed with the PSA-3A peptide than those targets pulsed with the PSA-3 peptide. This was observed when both concentration of peptide and effector to target cells ratios were titrated.
The experiments of this invention further show that T cells stimulated with dendritic cells (DCs) pulsed with PSA-3A peptide produced higher levels of IFN-γ than DCs pulsed with PSA-3 peptide. However, no increase in apoptosis was observed in T cells stimulated with the PSA-3A agonist as 15 compared with those stimulated with PSA-3. Notably, human T cell lines generated with the PSA-3A agonist had the ability to lyse human prostate carcinoma cells expressing native PSA in an MHC-A2 restricted manner.
In accordance with the present invention, recombinant vaccinia viruses were constructed containing the entire PSA transgene with or without the single amino acid change that constitutes the PSA-3A epitope. DCs infected with the recombinant vector containing the agonist amino acid change within the entire PSA gene (designated rV-PSA-3A) were more effective than the rV-PSA vector in enhancing IFN-γ production by T cells. Additionally, the PSA-3A agonist was shown to induce higher levels of T cell activation, as compared with the PSA-3 peptide, in an in vivo model using HLA-A2.1/Kb transgenic mice (strain reported by A. Vitiello et al., 1991, J. Exp. Med. 173:1007–1015; V. H. Engelhard et al., 1991, J. Immunol.6:1226–1232). Accordingly, the PSA-3A agonist epitope of this invention is useful for both peptide-mediated and vector-mediated immunotherapy protocols for prostate cancer.